circ_0091746以内源性竞争RNA方式结合miR-6848-5p在巨噬细胞抗新生隐球菌感染M1/M2极化中的作用及调控机制

Cryptococcosis is a kind of conditional fungi infection with high mortality due to therapy limitations. The pathogenesis is mainly related to the host's immune deficiency and the key factor is macrophage activation defect. The preliminary study found there was an abnormal expression profile of circRNAs in the peripheral blood of cryptococcal patients; circ_0091746 in macrophages was significantly higher and positively correlated with Arg1 which is the M2 polarized marker; Bioinformatics analysis revealed that there were multiple miR-6848-5p sequences for circ_0091746 and Arg1; overexpression of miR-6848-5p inhibited the expression of Arg1, which affected macrophage polarization. This project aims to study (1) the effects of circ_0091746 on macrophage function during Cryptococcus neoformans infection; (2) regulatory mechanism of circ_0091746 in M1/M2 polarization during Cryptococcus neoformans infection; (3) the regulation mechanism of macrophages circ_0091746 expression during Cryptococcus neoformans infection; (4) role of circ_0091746 on cryptococcosis process via cryptococcal mouse model. This study will provide a new theoretical basis for the immunotherapy of cryptococcosis.

隐球菌病是条件致病真菌导致的感染，治疗棘手，病死率高，致病机理主要与宿主免疫缺陷有关，其中巨噬细胞活化缺陷是关键因素之一。前期研究发现，隐球菌病患者外周血circRNA存在异常表达谱；巨噬细胞中高表达的circ_0091746与M2极化标志Arg1正相关；生物信息分析发现miR-6848-5p有多个circ_0091746和Arg1的结合位点；过表达miR-6848-5p可抑制Arg1表达，从而影响巨噬细胞极化状态。本项目拟进一步研究(1)circ_0091746对巨噬细胞抗新生隐球菌免疫反应中功能的影响；(2)circ_0091746对巨噬细胞抗新生隐球菌免疫反应中M1/M2极化的调控机制；(3)巨噬细胞抗新生隐球菌免疫反应中circ_0091746表达的调控机制；(4)利用隐球菌小鼠模型，研究circ_0091746对隐球菌病进程的影响。本研究将为隐球菌病的免疫疗法提供新的依据。

本研究按计划顺利完成，已发表SCI论文31篇，培养硕士研究生一名。研究主要结果如下：.1）circ_0091746对巨噬细胞抗新生隐球菌免疫应答中功能的影响。.si-circ_0091746可显著降低Arg-1 mRNA水平，增加NO、TNF-α、及miR-6848-5p水平；si-circ_0091746可增强巨噬细胞对隐球菌杀伤作用；828个基因在siRNA转染组存在差异性表达谱 （346个mRNA高表达，482个mRNA低表达）。转染circ_0091746 siRNA组中 native T细胞向Th1/Th17分化明显增强；上清液T细胞分泌细胞因子TNF-α和IFN-γ蛋白水平明显增加。. 2）circ_0091746对巨噬细胞抗新生隐球菌过程中M1/M2极化具体调控机制。.circ_0091746和miR-6848-5p在细胞胞浆内存在共定位关系。miR-6848-5p 水平在mimic组和inhibitor组分别上调约5倍、下降约0.3倍。转染miR-6848-5p后Arg1 3’UTR的荧光素活性明显下降约0.6倍。Arg1在miR-6848-5p mimic组中表达显著下降，在inhibitor组中表达显著上调。过表达circ_0091746组中TRAF2和IKK水平明显上调; miR-6848-5p inhibitor组中 TRAF2水平明显上调。共转染组的TRAF2 mRNA水平明显升高。.3）巨噬细胞抗新生隐球菌免疫反应中circ_0091746表达的表观遗传学调控机制。.隐球菌刺激组和未刺激组的circ_0091746基因启动子区CpG岛甲基化频率差异无统计学意义。RNA免疫共沉淀发现隐球菌感染后，巨噬细胞中HuR可与circ_0091746结合增高。隐球菌感染后过表达HuR组巨噬细胞中circ_0091746水平随时间明显增高，HuR干扰组巨噬细胞中circ_0091746水平随时间明显下降。.4）circ_0091746对隐球菌病疾病进程的影响。.经鼻吸入新生隐球菌悬液构建隐球菌感染小鼠模型。si-circ_0091746组小鼠的肺部和脑部菌落负荷明显增加，且生存期明显缩短；在7天、14天、21天si-circ_0091746组感染小鼠血清中TNF-a、IFN-g水平显著升高，IL-6无明显变化。